CN118006770A

CN118006770A - Detection of cancer using positive and negative strand differences of free DNA molecules

Info

Publication number: CN118006770A
Application number: CN202211409380.5A
Authority: CN
Inventors: 马士清; 叶可勇; 刘军; 陈一友
Original assignee: Hangzhou New Horizon Health Technology Co Ltd
Current assignee: Hangzhou New Horizon Health Technology Co Ltd
Priority date: 2022-11-09
Filing date: 2022-11-09
Publication date: 2024-05-10
Also published as: WO2024099301A1

Abstract

The present invention provides a method for analyzing the difference between the positive and negative strands of DNA in a sample for detecting cancer. The method comprises high throughput sequencing of sample DNA, and then calculating the imbalance (including but not limited to positive and negative strand relative depth, proportion, terminal sequence characteristics and mutation, etc.) of the positive and negative strands of the sample DNA based on the differences of the positive and negative strands in the sequencing data, and is used to detect cancer.

Description

Detection of cancer using positive and negative strand differences of free DNA molecules

Technical Field

The present invention relates to the field of biotechnology, in particular DNA detection. In particular, the invention relates to methods for detecting and analyzing plasma DNA. More particularly, the present invention relates to a method of calculating the difference in the number of positive and negative strands of DNA in a biological sample, and distinguishing whether an individual of origin of the biological sample has cancer, the type of cancer, the size of tumor, and the like based on this.

Background

Circulating free DNA refers to nucleic acid fragments that are free outside of the cell. Although clinical studies and applications concerning circulating free DNA are currently rapidly advancing, many molecular features associated with circulating free DNA remain to be explored, which is of great value for advancing the clinical application of analysis of circulating free DNA.

The study of tumor-derived DNA molecules in the plasma of cancer patients is a very challenging task because the tumor-derived DNA molecules are present in small amounts and cannot be easily distinguished from the normal-derived background DNA molecules. In the clinical research work currently existing, the most commonly used features are specific base mutations or nucleic acid modifications on DNA molecules, but it is difficult to accurately distinguish tumor-derived DNA from normal DNA due to random base mutation background on the whole genome and relatively few cancer-specific mutations. Particularly in the application fields of early cancer screening and diagnosis.

In the previous studies of circulating free DNA, a detection technique generally employed was a method of high throughput sequencing based on double strand library construction, but it is known that in circulating free DNA, there are cases where some DNA molecules exist with break damage or terminal protrusion, and some DNA molecules exist in single strand form as such. Since the first step of the commonly used double-stranded library construction method requires end repair of DNA molecules, and the adaptor ligation process is blunt-ended or TA ligation between double-stranded DNA molecules, the aforementioned special DNA molecules cannot be successfully constructed, and the information carried by the DNA molecules is lost.

Disclosure of Invention

It is an object of the present invention to provide a method for analyzing a sample for differences in positive and negative strands of DNA for detecting cancer. The method comprises high throughput sequencing of sample DNA, and then calculating the imbalance of positive and negative strands of the sample DNA from the differences of the positive and negative strands in the sequencing data, and for detecting cancer. For example, the method may be practiced to calculate the difference in the number of positive and negative strands in a region of the chromosome and compare it to a reference value for a known type of cancer, and when a sufficient number of regions have matching differences in the positive and negative strands, the type of cancer may be identified.

In some embodiments, single strand information of the DNA molecules in the sample may be retained by library construction of circulating free DNA using single strand DNA banking techniques. In some embodiments, an unbalanced signal of the number of positive and negative strands within a chromosomal region due to the presence of single stranded DNA may serve as a potential tumor signal. In some embodiments, the circulating free DNA (including single stranded DNA) is derived from tumor cells, and thus may also be referred to as circulating tumor DNA. In some embodiments, the sample contains both tumor DNA and non-tumor DNA.

For different types of cancers, different cancer-related molecular characteristics can be detected in the plasma of the corresponding patient, including but not limited to copy number variation, changes in methylation levels, single nucleotide mutations, viral sequence insertions, fragment size changes, terminal sequence characteristics, jagged terminal characteristics, chromosomal rearrangements, and the like.

In some embodiments, the differences in the positive and negative strands of the DNA fragments in the sample can be analyzed to infer the stage of cancer from which the sample originated. For example, if the positive and negative strand differences of the sample differ significantly from the values of the corresponding region of healthy controls, an early cancer may be indicated; and a smaller difference from the value of the cancer tissue sample may indicate advanced cancer.

In one aspect of the invention, there is provided a method of detecting cancer based on a biological sample obtained from an individual, comprising:

a) Determining the sequence and content of each of the single-stranded and double-stranded DNA molecules in the biological sample,

B) Determining the position of said DNA strand in the genome of said individual based on the results of a), and identifying that said DNA strand is the corresponding positive strand or negative strand of DNA in said genome,

C) Determining differential information of the positive strand of the DNA and the negative strand of the DNA of one or more specific regions in the genome,

D) Judging whether the individual suffers from the cancer or not according to the difference information.

In some embodiments, in step d), determining whether the individual has the cancer by comparing the DNA plus or minus strand difference information for each of the specific regions to corresponding DNA plus or minus strand difference information reference values for different types of cancer samples.

In some embodiments, the specific region of the genome is at least 50bp, at least 200bp, at least 500bp, at least 1000bp, at least 2000bp, at least 5000bp, at least 10000bp, or at least 20000bp in length.

In some embodiments, the specific region of the genome comprises a human chromosome 7 69,838,000bp to 69,848,000bp region. In some embodiments, the specific region of the genome comprises a region of human chromosome 7 69,838,000bp to 69,848,000bp that is at least 50bp, at least 200bp, at least 500bp, at least 1000bp, at least 2000bp, or at least 5000bp in length.

In some embodiments, in step c), at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 genome-specific regions are selected.

In some embodiments, the difference information is positive and negative strand relative coverage, DNA strand end sequences, DNA mutations, or any combination thereof.

In some embodiments, the difference information is the relative coverage of the positive and negative strands of DNA. In some embodiments, the relative coverage is calculated by (i) obtaining the number of fragments of the positive strand of DNA and the number of fragments of the negative strand of DNA for the particular region; (ii) Dividing the number of fragments of the positive strand of DNA by the number of fragments of the negative strand of DNA to obtain the relative coverage.

In some embodiments, the sequence and content of each DNA strand is determined in step a) using high throughput sequencing techniques. In some embodiments, the library construction approach used by the high throughput sequencing technique can distinguish between positive and negative strands of a DNA molecule. In some embodiments, the library construction means used is single stranded DNA library construction techniques.

In some embodiments, the method further comprises calculating a calibration function of the tumor size of the cancer and its DNA plus-minus strand difference information, thereby using the calibration function to determine the tumor size. In some embodiments, the calibration function is determined using pairs of data from reference samples of organisms with tumors of known size.

In some embodiments, the cancer is lung cancer, gastric cancer, liver cancer, esophageal cancer, breast cancer, cervical cancer, ovarian cancer, cervical cancer, prostate cancer, testicular cancer, colorectal cancer, colon cancer, pancreatic cancer, head and neck cancer, thyroid cancer, glioma, or skin cancer. In some embodiments, the cancer is hepatocellular carcinoma, colorectal cancer, esophageal cancer, or gastric cancer.

In some embodiments, the sample detected may be selected from the group consisting of tissue, whole blood, plasma, serum, urine, and stool. In some embodiments, the biological sample is blood, plasma, or serum.

In some embodiments, the single-stranded DNA molecules and double-stranded DNA molecules comprise plasma free DNA.

In one aspect of the present invention, there is provided a computer system characterized by being capable of determining the position of a single-stranded DNA molecule and a double-stranded DNA molecule in a biological sample in the genome of an individual based on the detection result of the sequence and the content of each DNA strand, and identifying whether the DNA strand is a corresponding positive strand or negative strand of DNA in the genome, and then determining difference information of the positive strand and the negative strand of DNA in one or more specific regions in the genome, and judging whether the individual suffers from the cancer based on the difference information.

In some embodiments, in order to achieve the above objective, the following technical solutions are adopted in the present invention:

A method of analyzing a biological sample of an organism, the biological sample comprising DNA molecules derived from normal cells and possibly from cells associated with cancer, wherein at least a portion of the nucleic acid molecules are present in the biological sample in a free state. The detection method comprises the following steps:

1. Detecting DNA molecules in a biological sample, wherein the detection method is selected to distinguish between positive and negative strands of the DNA molecules;

2. Identifying the location of the DNA molecule in a reference genome of the organism based on the detection result; based on the identified locations, nucleic acid molecules of the respective samples are identified as being from the chromosomal region.

3. Differential information of the number of positive and negative strands of all or part of the region on the chromosome is calculated, and whether the region of the chromosome exhibits positive and negative strand imbalance is determined based on the calculated value and the reference baseline value.

By using the method for detecting the difference of the positive and negative chains of the DNA molecules in the sample, the positive and negative chain difference signals of the DNA of the sample can be effectively calculated, and the cancer type or tumor size information corresponding to the source of the sample can be indicated by comparing the calculated sample signals with the signals of the reference samples of known cancer types or tumors of known sizes.

According to yet another aspect of the present invention, a system for determining positive and negative strand differential information of DNA molecules in a sample is provided. According to an embodiment of the invention, the system comprises: a sequencing library construction device, said sequencing library construction device being according to claim 3; the sequencing device is connected with the sequencing library construction device so as to sequence the sequencing library of the sample and obtain a sequencing result of the sample; and the analysis device is used for analyzing the sequencing result of the sample so as to obtain the positive and negative strand difference information of the DNA molecules.

The system for determining the positive and negative strand difference information of the DNA molecules can sensitively, accurately and efficiently determine the positive and negative strand difference information of the DNA molecules in a trace sample, and can be used for distinguishing cancers.

Additional aspects and advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention.

Drawings

The foregoing and/or additional aspects and advantages of the invention will become apparent and may be better understood from the following description of embodiments taken in conjunction with the accompanying drawings in which:

FIG. 1 shows a flow chart for detecting and analyzing differences in the positive and negative strands of DNA molecules in a sample according to one embodiment of the invention;

FIG. 2 shows differences in positive and negative strand signals of blood free DNA molecules of a cancer sample and blood free DNA molecules of a healthy control sample in the same chromosome region, wherein the cancer sample is red, the healthy control sample is yellow, the human chromosome 7 region coordinates are on the abscissa, and the positive and negative strand fragment number deviation values are on the ordinate, detected in accordance with an embodiment of the present invention.

Detailed Description

Reference will now be made in detail to embodiments of the present invention, examples of which are illustrated in the accompanying drawings. Those skilled in the art will appreciate that the embodiments described below by referring to the drawings are exemplary only for the purpose of illustrating the invention and are not to be construed as limiting the invention. The examples, without specific technical terms being noted, are conventional products commercially available, such as NEB, etc., according to the techniques or conditions described in the literature in this field or according to product specifications.

According to one aspect of the present invention, there is provided a method of analyzing positive and negative strand differential information of a DNA molecule in a sample. Referring to fig. 1, according to an embodiment of the present invention, the method may include the steps of:

1. The biological sample is extracted using a suitable DNA extraction kit.

2. DNA samples for library construction were first quantitated using Qubit 4.0 and quantitated using 1*Qubit dsDNAHS assay kit. The DNA used for library construction may be used in an initial amount of 0.5-20 ng.

4. Dephosphorylation and high temperature denaturation.

The DNA sample is dephosphorylated, the reaction system comprises alkaline phosphatase and a reaction buffer, the reaction is carried out for 30 minutes at 37 ℃, then the reaction is carried out for 5 minutes at 95 ℃, and the DNA is denatured into single strands at high temperature.

The single-stranded adaptor is connected with the single-stranded DNA product in the last step, and the connection is between two single-stranded DNA molecules.

The 5-1 single-stranded linker sequence is 5'-Pho- (N) _n AAGTCGGATCGTAGCCATG-3' ddC (SEQ ID NO: 1), the 5 '-end of the linker sequence is phosphorylated for modification and the 3' -end is dideoxy for modification, thus preventing self-ligation of the linker and self-ligation between DNA molecules in the ligation reaction. And at the same time, the random sequence of n bases of the primer at the 5' end of the joint is used for correcting PCR amplification errors possibly caused during low initial quantity library establishment, and n is a positive integer between 1 and 15.

5-2 The ligation reaction system comprises: denatured single-stranded DNA product, single-stranded linker, PEG8000, ligase buffer and T4 RNA ligase.

The reaction conditions of 5-3 were either 16℃overnight or 30℃for 2 hours.

6 Extension reaction. And (3) taking the single-stranded connection product as a template, designing a primer by using a known sequence of the single-stranded joint, and extending to obtain a double-stranded DNA product.

The sequence of the 6-1 primer is as follows: CAACTCCTTGGCTCACAGAACGACATGGCTACGATCCGACTT (SEQ ID NO: 2).

The 6-2 reaction system comprises: single stranded DNA ligation products, primers, DNA polymerase buffer and DNA polymerase.

6-3 Reaction conditions: 95℃for 2min, 45℃for 30 sec, 72℃for 5min.

7. The double-end connector is connected. The step is an intermolecular ligation between the double-stranded DNA product produced in the previous step and the double-stranded DNA adaptor.

The 7-1 double-stranded DNA linker sequence is:

F sequence: 5'-Pho-GAAGTCGGAGGCCAAGCGGTCTTAGGAAGACAA-3' (SEQ ID NO: 3).

R sequence: 5'-CAACTCCTTGGCTCACAGAACGACATGGCTACGATCCGACTTCT-3' (SEQ ID NO: 4).

The R sequence is complementary to the F sequence in direction, and specifically, the last nucleotide T of the R sequence is a prominent nucleotide T.

The 7-2 ligation reaction system comprises: the double-stranded DNA product of the previous step, double-stranded DNA linker, T4 DNALIGASE and T4 DNA LIGASE buffer.

The reaction conditions of 7-3 were 20℃for 30 minutes.

PCR amplification. The ligation product obtained in the above step is used as a template, and the known sequences of the joints at the two ends of the 5 'end and the 3' end are used as forward and reverse primers for PCR amplification.

8-1 Forward sequence:

5’-GCATGGCGACCTTATCAGNNNNNNNNNTTGTCTTCCTAAGACCGCTTGG-3’(SEQ ID NO:5)。

reverse sequence:

5’-Pho-CTCTCAGTACGTCAGCAGTTNNNNNNNNNNCAACTCCTTGGCTCACAGAAC-3’(SEQ ID NO:6).

Of which 10 random bases are used to distinguish between different samples in high throughput sequencing.

The 8-2 reaction system comprises: the product of the last step, forward and reverse PCR primer, high-fidelity DNA polymerase and DNA polymerase buffer.

8-3 Reaction conditions: denaturation at 98℃for 2min;98 ℃,15s,60 ℃,30s,72 ℃ and 30s. The process is carried out for 8-15 cycles. Then, the reaction was carried out at 72℃for 5min.

9. And (3) purifying the PCR product by a magnetic bead method to obtain a target product, and recovering to obtain a library.

10. And (5) performing on-machine sequencing on the library to obtain sequencing data.

11. Sequencing data analysis. And (3) performing quality control on the sequencing data obtained in the last step by using FastQC software, comparing the sequencing sequence with a standard human genome sequence by using comparison software BWA to obtain information positioned at the corresponding position of the human genome, namely generating a SAM format file, and finally obtaining a deviation value of the number of positive and negative chain fragments by using SAMtools software according to the comparison label in the SAM file (figure 2).

The results show that the blood free DNA molecules of the cancer samples (red line) and the blood free DNA molecules of the healthy control samples (yellow line) have significant differences in the positive and negative strand signals of the same chromosomal region.

The foregoing is a further detailed description of the invention in connection with specific embodiments, and is not intended to limit the practice of the invention to such descriptions. Those skilled in the art will appreciate that: many changes, modifications, substitutions and variations may be made to the embodiments without departing from the spirit and principles of the invention, the scope of which is defined by the claims and their equivalents.

FastQC, BWA, SAMtools is a software name, and is a Chinese name without industry in China, and is directly described by English or abbreviation.

Claims

1.A method of detecting cancer based on a biological sample obtained from an individual, comprising:

a) Determining the sequence and content of each of the single-stranded DNA molecules and double-stranded DNA molecules in the biological sample;

b) Determining the position of the DNA strand in the genome of the individual from the result of a) and identifying the DNA strand as a corresponding positive strand or negative strand of DNA in the genome;

c) Determining differential information of the positive strand of DNA and the negative strand of DNA for one or more specific regions in the genome;

d) And judging whether the individual suffers from the cancer or not according to the difference information.

2. The method according to claim 1, wherein in step d) it is determined whether the individual has the cancer by comparing the DNA plus or minus strand difference information of each of the specific regions with corresponding DNA plus or minus strand difference information reference values of different types of cancer samples.

3. The method of claim 1 or 2, wherein the specific region of the genome is at least 50bp, at least 200bp, at least 500bp, at least 1000bp, at least 2000bp, at least 5000bp, at least 10000bp, or at least 20000bp in length.

4. A method according to any one of claims 1-3, wherein in step c) at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 of said specific areas are selected.

5. The method of any one of claims 1-4, wherein the differential information is positive and negative strand relative coverage, DNA strand end sequences, DNA mutations, or any combination thereof.

6. The method of claim 5, wherein the difference information is the relative coverage of the positive and negative strands of DNA calculated by (i) obtaining the number of fragments of the positive strand of DNA and the number of fragments of the negative strand of DNA for the specific region; (ii) Dividing the number of fragments of the positive strand of DNA by the number of fragments of the negative strand of DNA to obtain the relative coverage.

7. The method according to any one of claims 1 to 6, wherein the sequence and content of each DNA strand is determined in step a) using high throughput sequencing techniques; preferably, the library construction method used by the high throughput sequencing technology can distinguish between the positive and negative strands of a DNA molecule.

8. The method according to any one of claims 1 to 7, wherein the library construction means used is single stranded DNA library construction technology.

9. The method of any one of claims 1-8, further comprising calculating a calibration function of the size of the tumor of the cancer and its DNA plus-minus strand difference information, thereby using the calibration function to determine the size of the tumor.

10. The method of claim 9, wherein the calibration function is determined using pairs of data from a reference sample of an organism having a tumor of known size.

11. The method of any one of claims 1-10, wherein the cancer is hepatocellular carcinoma, colorectal cancer, esophageal cancer, or gastric cancer.

12. The method of any one of claims 1-11, wherein the biological sample is blood, plasma, or serum.

13. The method of any one of claims 1-12, wherein the single-stranded DNA molecule and double-stranded DNA molecule comprise plasma free DNA.

14. A computer system, characterized in that steps b) -d) of the method according to any of claims 1-13 are enabled.